Methods of inducing IL-10 production

ABSTRACT

The invention provides a method of inducing IL-10 production.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/010,859, filed Dec. 13, 2004, which issued as U.S. Pat. No.7,160,861, which claims the benefit of U.S. Provisional PatentApplication No. 60/529,137, filed Dec. 12, 2003 and U.S. ProvisionalPatent Application No. 60/571,145, filed May 14, 2004, each of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods of inducing interleukin-10 productionin a cell.

BACKGROUND OF THE INVENTION

LGALS1 belongs to the family of animal lectins, which are highlyconserved throughout evolution. Galectins share remarkable sequencesimilarities in the carbohydrate recognition domain and have affinityfor polylactosamine-enriched glycoconjugates (1,2). LGALS1 encodes aβ-galactoside binding protein (β-GBP) and is known to bind to leukocytemembrane antigens like CD45, CD43, and CD7 (3-5). The protein can occurboth as a monomer (˜14.5 kDa, β-GBP) and a homodimer (˜29 kDa,galectin-1) (2,6). In its monomeric form, β-GBP inhibits T cellproliferation by arresting cells in the S and G₂/M phases (7). As ahomodimer, galectin-1 has various effects on cell-cell and cell-matrixinteractions (8,9). By crosslinking of T-cell surface proteins,galectin-1 induces apoptosis of activated but not resting T-cells (10).This process is mediated by activation of transcription factors NFAT andAP-1 and by downregulation of Bcl-2 protein (11,12). Susceptibility ofimmature thymocytes to galectin-1 induced apoptosis suggests a role forthe protein during thymic selection (13).

One of the most intriguing functions of the galectin-1 protein is itsrole in immunomodulation. Research over the past decades has identifieda beneficial role for galectin-1 in several models of autoimmunediseases (14-16). Recently, it was demonstrated that galectin-1 inhibitsthe allogeneic T cell response in a dose- and carbohydrate dependentway. Downregulation of the immune response appeared to involve bothapoptosis of activated T cells and non-apoptotic mechanisms (17).Galectin-1 can play a role in downregulation of the immune response bymodulating cytokine production. In a mouse model for rheumatoidarthritis, treatment with galectin-1 protein resulted in a shift from aT helper 1 (TH1) toward a T helper 2 (TH2) type response, which wasaccompanied by downregulation of interferon (IFN)-γ and upregulation ofinterleukin (IL)-5 in draining lymph nodes (16). Furthermore, galectin-1treatment induced a reduction of IFN-γ and tumor necrosis factor (TNF)-αproduction in con-A induced hepatitis in mice (18) and in IL-2 activatedT cells (9). Recently, it was described that galectin-1 also suppressesexperimental colitis in mice (19). In this model, a decrease ininflammatory cytokine production was observed, together with apoptosisof mononuclear cells.

SUMMARY OF THE INVENTION

The invention is based on the discovery that homodimeric galectin-1induces high levels of IL-10 and IL-1β production in both activated andresting T-cells. Accordingly, the invention features methods of inducingIL-10 or IL-1β production in a cell or a bodily tissue. IL-10 or IL-1βproduction is induced by contacting a cell or a tissue a multimericgalectin-1 polypeptide. The multimeric galectin-1 polypeptide is adimer. The multimeric galectin-1 polypeptide is stable. By stable ismeant that the multimer does not dissociate to the monomeric form at lowconcentrations (e.g., less than 7 μM) Exemplary galectin-1 polypeptidesinclude the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. The cellis any cell that is capable of expressing IL-10, e.g., a lymphocyte suchas a T-cell, B-cell or monocyte. The T-cell is activated. Alternatively,the T-cell is non-activated. Optionally, the T cell is CD4 and/or CD8positive. The cell is contacted in vivo, in vitro, or ex vivo.

The invention also features methods of preventing or alleviating asymptom of an immune mediated disorder in a subject by identifying asubject suffering from or at risk of developing an immune mediateddisorder and administering to the subject a multimeric galectin-1polypeptide. The immune mediated disorder is for example, asthma, aninflammatory bowel disease such as Crohn's disease or ulcerative colitisor an autoimmune disease such as multiple sclerosis, diabetes,rheumatoid arthritis, or systemic lupus erthematosis, transplantrejection or food related allergies.

Inflammation is inhibited by administering to an inflamed tissue (or atissue that is at risk of becoming inflamed) a multimeric galectin-1polypeptide. An inflamed tissue is characterized by redness, pain andswelling of the tissue. The tissue includes epithelial tissue, pulmonarytissue, nervous tissue pancreatic tissue or liver tissue. For example,the epithelial tissue is intestinal tissue or skin.

The subject is suffering from or at risk of developing an inflammatorydisorder. Inflammatory disorders include for example, cardiovascularinflammation, gastrointestinal inflammation, hepatic inflammation,pulmonary inflammation, autoimmune disorders, allergy or skeletalinflammation. A subject suffering from or at risk of developing aninflammatory disorder is identified by methods known in the art, e.g.,gross examination of tissue or detection of inflammation associatedmarkers in tissue or blood. Symptoms of inflammation include pain,redness and swelling of the affected tissue. A subject suffering fromgastrointestinal inflammation, such as colitis, is identifiedhistologically by the presence of mucosal necrosis or hemorrhagiclesions in the colon, frequent diarrhea or blood and pus in the stool.

The invention further features methods of increasing graft survival,e.g., allograft by administering to a subject a multimeric galectin-1polypeptide. The composition is administered to the subject prior to,concomitantly with or after the subject receives the transplant.Optionally, the composition is administered over a pre-selected periodof time such as 1-2 weeks.

The invention also features a method of inducing apoptosis by contactinga cell will a multimeric galectin-1 polypeptide in an amount sufficientto induce apoptosis. The multimeric galectin-polypeptide induces at alower concentration than wild-type, e.g., monomeric galectin. Forexample, galectin is induced at concentrations less than 20 μM. The cellcan be provided in vitro, in vivo or ex vivo. The cell is any cell whereinduction of apoptosis is desired such as a cancer cell.

Also included in the invention is a purified vaccine compositioncontaining a galectin-1 polypeptide and an antigen. Methods ofvaccination using the vaccine compositions of the invention is alsoprovided. A subject is vaccinated a by administering to the subject afirst composition containing a galectin-1 polypeptide and a secondcomposition containing an antigen. The first composition is administeredconcomitantly with the second composition. Alternatively, the firstcomposition in administered prior to or after the second composition.

The subject is a mammal such as human, a primate, mouse, rat, dog, cat,cow, horse, or pig.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 are bar charts depicting that galectin-1 stimulation highlyinduces IL-10 production and decreases IFN-γ production in humanperipheral blood mononuclear cells and CD4⁺ and CD8⁺T-lymphocytes. A,IL-10 protein production in anti-CD3 activated PBMC's using differentconcentrations of galectin-1 protein (20 μM, 2 μM and 0.2 μM) and theeffect of inhibitors (0.1M lactose or anti-galectin-1 antibodies). B,IL-10 protein production in CD4⁺ and CD8⁺ T-lymphocytes. The result ofone representative donor (donor #1) out of four is shown. C, IFN-γprotein production in CD4⁺ and CD8⁺ T-lymphocytes. The relative IFN-γproduction (anti-CD3/CD28 versus anti-CD3/CD28 plus galectin-1) is shownas mean ±S.D. of four independent donors. D, IL-10 mRNA production inCD4⁺ and CD8⁺T-lymphocytes as measured by real-time PCR analysis. Theresult of one out of four donors (donor #1) is shown as the factordifference relative to a common calibrator, after normalization to ahousekeeping gene. The results of all four donors are given in table 1.E, CD25 and CD69 expression as determined by FACS analysis. Total PBMC'swere left resting or stimulated for 24 hrs as indicated, beforeanalyzing expression of CD25 and CD69.

FIG. 2A is a photograph of a gel showing semi-quantitative RT-PCRanalysis for LGALS1, IL-10, IFN-γ, and GAPDH. Case numbers are aspreviously reported (21) and the number of PCR cycles used for theamplification are shown between parentheses.

FIG. 2B is a bar chart showing real-time PCR analysis for LGALS1, IL-10,and IFN-γ. Case numbers are the same as shown in Table 1. Left y-axis:relative LGALS1 mRNA quantity; right y-axis: relative IL-10 andIFN-γmRNA quantity.

FIG. 3 are photographs showing galectin-1 protein expression inrejecting kidney allografts and in normal control tissue. In normalkidney, galectin-1 is present in glomerular mesangial epithelial cells(A), smooth muscle cells of large vessels (B) and occasionally cells ininterstitium are galectin-1 positive (C), whereas no galectin-1 isobserved in endothelial cells from peritubular capillaries (C). Inrejecting kidney allografts (D-F), galectin-1 is highly upregulated inendothelial cells from large vessels (D,E), and in endothelial cellsfrom peritubular capillaries in interstitium (F). Originalmagnifications A-D: 400×, E: 630×, F: 400×.

FIG. 4 is a bar chart comparing IL-10 induction by stable galectin-1dimers compared to wild-type galectin-1. Low concentrations of stablegalectin-1 homodimers highly induce IL-10 production in total PBMC's.Wild-type galectin-1 was tested at four different concentrations (20,10, 1, and 0.1 uM). Stable galectin-1 homodimers (construct 1 andconstruct 2) were tested at low concentrations (1 uM and 0.1 uM), thatwere not effective for wild-type galectin-1. Stable galectin-1homodimers induce a high IL-10 production at concentrations up to200-fold lower as compared to wild-type galectin.

FIG. 5 is a bar chart showing the a comparison of IL-10 production bywild type (mGal) Galectin-1 at 3 concentrations (20, 2 and 0.2 μM) anddimeric (dGal) Galectin-1 at 3 concentrations (2, 1 and 0.2 μM) in CD3activated lymphocytes of a representative donor.

FIG. 6 is a bar chart showing IL-10 production of lymphocytes afterincubation with dimeric Galectin-1 (1 μM) is specifically blocked bypre-incubation with lactose (0.1M).

FIG. 7 is a bar chart showing the effect of wildtype galectin-1 andstable galectin-1 homodimers on apoptosis induction in MOLT-4 T-cells.Results are shown as the percentage of AnnexinV positive cells minuspercentage of AnnexinV positive cells in unstimulated control cells. Theerror bars represent the standard deviation of four independentexperiments.

FIG. 8 is a bar chart showing IL-10 production by the lymphocytes of 5different volunteers after 24 hours of incubation with CD3 (1 ng/ml) and3 different concentrations of dimeric Galectin-1 (2, 1, 0.2 μM).

FIG. 9 is a bar chart showing IL-10 mRNA is expressed in the lymphocytesof 5 different volunteers after 24 hrs. Lymphocytes were incubated withCD3 (1 ng/ml) and 3 concentrations of dimeric Galectin-1 (2, 1 and 0.2μM).

FIG. 10 is a bar chart showing the comparison of IL-1β production bywild type (mGal) Galectin-1 at 3 concentrations (20, 2 and 0.2 μM) anddimeric (dGal) Galectin-1 at 3 concentrations (2, 1 and 0.2 μM) in CD3activated lymphocytes of a representative donor.

FIG. 11 is a bar chart showing IL-1β production by lymphocytes of 5different volunteers after 24 hours of incubation with CD3 (1 ng/ml) and3 different concentrations of dimeric Galectin-1 (2, 1, 0.2 μM).

FIG. 12 is a bar chart showing IL-1 beta production of lymphocytes afterincubation with dimeric Galectin-1 (1 μM) is specifically blocked bypreincubation with lactose (0.1M).

FIG. 13 is a bar chart showing IL-10 mRNA expressed in biopsies of apatient with colitis. Biopsies, were taken at 2 locations, control (notinflamed) and inflamed tissue were incubated in tissue culture mediumfor 8 hours with or without 2 different concentrations of dimericGalectin-1 (2.5 and 0.5 μM).

DETAILED DESCRIPTION

The invention is based in part on the unexpected discovery that a stablehomodimeric galectin-1 induces high levels of IL-10 production in bothactivated and resting T-cells. In contrast, monomeric galectin-1 (i.e.,B-galactoside binding protein) does not induce IL-10 production.

Galectin-1 Polypeptides

Galectins are defined as lectins having both galactoside-binding abilityand a characteristic amino acid sequence. Galectin-1 is a homodimericlectin with specificity for beta-galactosides. The lectin is synthesizedin the cytosol of mammalian cells, where it accumulates in a monomericform, as it is secreted from the cell where it forms homodimers.Typically, the functional lectins exist in monomer-dimer equilibriumwith a K of 7 μM and the equilibrium rate is rather slow (t_(1/2)=10hrs).

The present invention provides galactin-1 multimers (e.g. dimers,trimers, etc.) Preferably, the galectin-1 multimer is a homodimer. Thegalectin-1 multimers are more stable than wild-type galectin-1 andinduce IL-10 production. The galectin-1 multimer further induces IL-1βproduction. By stable is meant that the galectin-1 multimers do notdissociate to monomers at low concentrations. For example, thegalectin-1 polypeptides of eth invention are multimers at concentrationslower than 7 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, 0.1 μM or 0.001 μM.The galectin multimers are effective at inducing IL-10 and IL-1βproduction at lower concentrations than wild-type galectin. The stablegalectin dimers induce IL-10 production at 10, 50, 100 150, 200, 250,300 fold lower concentrations than wild type galectin. Thus, thegalectin-1 polypeptides of the invention have advantages to wild-typegalectin as they can be administered at lower concentration andtherefore avoiding the development of undesired side effects. In afurther embodiment, the galectin-1 multimers induce apoptosis, e.g.,programmed cell death. In comparison to direct IL-10 treatment andgalectin-1 monomer treatment, stable galectin-1 homodimers have theadvantage of effectively eliminating activated T cells by induction ofapoptosis. In another embodiment the galectin-1 multimers down-regulate,e.g., inhibit IFNγ production

Stable galectin multimers are produced by constructing a recombinantgalectin-1 monomer with a leucine zipper moiety on the N-terminus and/orC-terminus of a wild type (normal) galectin polypeptide, variant, orfragment thereof. Optionally, a hinge region joins the wild-typegalacetin-1 moiety and the leucine zipper moiety. Stable galectinmultimers are easily prepared using modern cloning techniques, or may besynthesized by solid-state methods. Alternative to recombinantexpression, galectin multimers peptides can be synthesized chemicallyusing standard peptide synthesis techniques. Stable dimers also areconstructed using known protein engineering techniques, such as computerassisted rational design.

Suitable sources of nucleic acids encoding wild-type galectin-1polypeptide include for example the human galectin-1 nucleic acids (andthe encoded protein sequences) available as GenBank Accession No.BT006775 and AAP35421 respectively and GenBank Accession No. BT007914and AAP36586 respectively and are incorporated herein by reference intheir entirety.

Exemplary recombinant galectin-1 monomers useful for producing thestable multimer includes the amino acid sequence of SEQ ID NO: 1 or 2,variants or fragments thereof as shown below. SEQ ID NO:1 wasconstructed with a histidine tagged leucine zipper moiety (italic)linked to the amino terminus of a wild type galectin-1 moiety(underlined) with a hinge spacer moiety (bold). Leucine residues in theFOS zipper are indicated in bold SEQ ID NO:2 was constructed with ahistidine tagged leucine zipper moiety (italic) linked to the carboxylterminus of a wild type galectin-1 moiety (underlined) with a hingespacer moiety (bold)

ProtFOSHingeGAL1 (SEQ ID NO:1) MGSSHHHHHHSSGLVPRGSHMCGG

TDT

QAETDRLEDEKSA

QTEIAN

LKEKEK

EFILAAHGGC PKPSTPPGSSH MACGLVASNLNLKPGECLRVRGEVAPDAKSFVLNLGKDSNNLCLHFNPRFNAHGDANTIVCNSKDGGAWGTEQREAVFPFQPGSVAEVCITFDQANLTVKLPDGYEFKFPNRLNLEAINYMA ADGDFKIKCVAFDGProtGAL1HingeFOS (SEQ ID NO:2) MGSSHHHHHHSSGLVPRGSHMACGLVASNLNLKPGECLRVRGEVAPDAKSFVLNLGKDSNNLCLHFNPRFNAHGDANTIVCNSKDGGAWGTEQREAVFPFQPGSVAEVCITFDQANLTVKLPDGYEFKFPNRLNLEAINYMAADGDFKIK CVAFDG SPKPSTPPGCSCGGLTDTLQAETDRLEDEKSALQTEIANLLKEK EKLEFILAAHGGT

The galectin-1 multimer are polymers of L-amino acids, D-amino acids, ora combination of both. For example, in various embodiments, the peptidesare D retro-inverso peptides. The term “retro-inverso isomer” refers toan isomer of a linear peptide in which the direction of the sequence isreversed and the chirality of each amino acid residue is inverted. See,e.g., Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature,368, 692-693 (1994). The net result of combining D-enantiomers andreverse synthesis is that the positions of carbonyl and amino groups ineach amide bond are exchanged, while the position of the side-chaingroups at each alpha carbon is preserved. Unless specifically statedotherwise, it is presumed that any given L-amino acid sequence of theinvention may be made into a D retro-inverso peptide by synthesizing areverse of the sequence for the corresponding native L-amino acidsequence.

The recombinant galectin-1 monomers or galectin-1 variants are used toproduce chimeric or fusion proteins. As used herein, a galectin-1“chimeric protein” or “fusion protein” comprises a recombinantgalectin-1 monomer (e.g., SEQ ID NO: 1 or SEQ ID NO:2) operativelylinked to a second polypeptide. Optionally, the fusion proteins are usedto form stable multimers such as dimers.

For example, in one aspect the invention provides a chimeric peptidethat include a first domain containing recombinant galectin-1 monomeroperably linked to a second domain containing a translocation sequence.

A “translocation sequence” refers to any sequence of amino acids thatdirects a peptide in which it is present to a desired cellulardestination. For example the translocation sequence is polyarginine.Thus, the translocation sequence can direct or facilitate penetration ofthe peptide across a biological membrane, e.g., a phospholipid membrane,mitochondrial membrane, or nuclear membrane. For example thetranslocation sequence directs the peptide from outside the cell,through the plasma membrane, and into the cytoplasm or to a desiredlocation within the cell, e.g., the nucleus, the ribosome, themitochondria, the ER, a lysosome, or peroxisome. Alternatively, or inaddition, the translocation sequence can direct the peptide across aphysiological barrier such as the blood-brain barrier, the trans-mucosalbarrier, or the hematoencephalic, hematoretinal, gastrointestinal andpulmonary barriers.

In another embodiment, the fusion protein is a GST-recombinantgalectin-1 monomer peptide fusion protein in which the recombinantgalectin-1 monomer sequence is fused to the C-terminus of the GST (i.e.,glutathione S-transferase) sequence. Such fusion proteins can facilitatethe purification of recombinant galectin-1 peptide.

In another embodiment, the fusion protein is a recombinant galectin-1monomer -immunoglobulin fusion protein in which the recombinantgalectin-1 monomer sequences are fused to sequences derived from amember of the immunoglobulin protein family.

Also included in the invention are derivatives, fragments, homologs,analogs and variants of the galectin-1 multimer polypeptide and nucleicacids encoding these polypeptide. For nucleic acids, derivatives,fragments, and analogs provided herein are defined as sequences of atleast 6 (contiguous) nucleic acids, and which have a length sufficientto allow for specific hybridization. For amino acids, derivatives,fragments, and analogs provided herein are defined as sequences of atleast 4 (contiguous) amino acids, a length sufficient to allow forspecific recognition of an epitope.

The length of the fragments are less than the length of thecorresponding full-length nucleic acid or polypeptide from which thegalectin-1 multimer polypeptide, or nucleic acid encoding same, isderived. Derivatives and analogs may be full length or other than fulllength, if the derivative or analog contains a modified nucleic acid oramino acid. Derivatives or analogs of the galectin-1 multimerpolypeptide include, e.g., molecules including regions that aresubstantially homologous to the peptides, in various embodiments, by atleast about 30%, 50%, 70%, 80%, or 95%, 98%, or even 99%, identity overan amino acid sequence of identical size or when compared to an alignedsequence in which the alignment is done by a computer homology programknown in the art. For example sequence identity can be measured usingsequence analysis software (Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705), with the defaultparameters therein.

In the case of polypeptide sequences, which are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thus,included in the invention are peptides having mutated sequences suchthat they remain homologous, e.g. in sequence, in function, and inantigenic character or other function, with a protein having thecorresponding parent sequence. Such mutations can, for example, bemutations involving conservative amino acid changes, e.g., changesbetween amino acids of broadly similar molecular properties. Forexample, interchanges within the aliphatic group alanine, valine,leucine and isoleucine can be considered as conservative. Sometimessubstitution of glycine for one of these can also be consideredconservative. Other conservative interchanges include those within thealiphatic group aspartate and glutamate; within the amide groupasparagine and glutamine; within the hydroxyl group serine andthreonine; within the aromatic group phenylalanine, tyrosine andtryptophan; within the basic group lysine, arginine and histidine; andwithin the sulfur-containing group methionine and cysteine. Sometimessubstitution within the group methionine and leucine can also beconsidered conservative. Preferred conservative substitution groups areaspartate-glutamate; asparagine-glutamine; valine-leucine-isoleucine;alanine-valine; phenylalanine-tyrosine; and lysine-arginine.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide, which is 50% identical to the referencepolypeptide over its entire length. Of course, other polypeptides willmeet the same criteria.

The invention also encompasses allelic variants of the disclosedpolynucleotides or peptides; that is, naturally-occurring alternativeforms of the isolated polynucleotide that also encode peptides that areidentical, homologous or related to that encoded by the polynucleotides.Alternatively, non-naturally occurring variants may be produced bymutagenesis techniques or by direct synthesis.

Species homologs of the disclosed polynucleotides and peptides are alsoprovided by the present invention. “Variant” refers to a polynucleotideor polypeptide differing from the polynucleotide or polypeptide of thepresent invention, but retaining essential properties thereof.Generally, variants are overall closely similar, and in many regions,identical to the polynucleotide or polypeptide of the present invention.The variants may contain alterations in the coding regions, non-codingregions, or both.

In some embodiments, altered sequences include insertions such that theoverall amino acid sequence is lengthened while the protein retainsIL-10 inducing properties. Additionally, altered sequences may includerandom or designed internal deletions that shorten the overall aminoacid sequence while the protein retains transport properties. Thealtered sequences can additionally or alternatively be encoded bypolynucleotides that hybridize under stringent conditions with theappropriate strand of the naturally-occurring polynucleotide encoding apolypeptide or peptide from which the galectin-1 multimer polypeptide isderived. The variant peptide can be tested for IL-10 induction using theherein described assays. ‘Stringent conditions’ are sequence dependentand will be different in different circumstances. Generally, stringentconditions can be selected to be about 5° C. lower than the thermalmelting point (T_(M)) for the specific sequence at a defined ionicstrength and pH. The T_(M) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Typically, stringent conditions will be thosein which the salt concentration is at least about 0.02 molar at pH 7 andthe temperature is at least about 60° C. As other factors may affect thestringency of hybridization (including, among others, base compositionand size of the complementary strands), the presence of organic solventsand the extent of base mismatching, the combination of parameters ismore important than the absolute measure of any one.

High stringency can include, e.g., Step 1: Filters containing DNA arepretreated for 8 hours to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Step 2: Filters arehybridized for 48 hours at 65° C. in the above prehybridization mixtureto which is added 100 mg/ml denatured salmon sperm DNA and 5-20×10⁶ cpmof ³²P-labeled probe. Step 3: Filters are washed for 1 hour at 37° C. ina solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes. Step 4:Filters are autoradiographed. Other conditions of high stringency thatmay be used are well known in the art. See, e.g., Ausubel et al.,(eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley andSons, NY; and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORYMANNUAL, Stockton Press, NY.

Moderate stringency conditions can include the following: Step 1:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100mg/ml denatured salmon sperm DNA. Step 2: Filters are hybridized for18-20 hours at 55° C. in the same solution with 5-20×10⁶ cpm ³²P-labeledprobe added. Step 3: Filters are washed at 37° C. for 1 hour in asolution containing 2×SSC, 0.1% SDS, then washed twice for 30 minutes at60° C. in a solution containing 1×SSC and 0.1% SDS. Step 4: Filters areblotted dry and exposed for autoradiography. Other conditions ofmoderate stringency that may be used are well-known in the art. See,e.g., Ausubel et al., (eds.), 1993, CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley and Sons, NY; and Kriegler, 1990, GENE TRANSFER ANDEXPRESSION, A LABORATORY MANNUAL, Stockton Press, NY.

Low stringency can include: Step 1: Filters containing DNA arepretreated for 6 hours at 40° C. in a solution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Step 2: Filters arehybridized for 18-20 hours at 40° C. in the same solution with theaddition of 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon spermDNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm ³²P-labeled probe.Step 3: Filters are washed for 1.5 hours at 55° C. in a solutioncontaining 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. Thewash solution is replaced with fresh solution and incubated anadditional 1.5 hours at 60° C. Step 4: Filters are blotted dry andexposed for autoradiography. If necessary, filters are washed for athird time at 65-68° C. and reexposed to film. Other conditions of lowstringency that may be used are well known in the art (e.g., as employedfor cross-species hybridizations). See, e.g., Ausubel et al., (eds.),1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, NY;and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANNUAL,Stockton Press, NY.

Methods of Inducing IL-10 Production

Interleukin-10 (IL-10) is an essential negative regulator of theinflammatory response. IL-10 inhibits activation and effector functionof T cells, monocytes, and macrophages. Il-10 is a multifunctionalcytokine with diverse effects on most hemopoietic cell types. Theprincipal function of IL-10 is to limit and ultimately terminateinflammatory responses. In addition to these activities, IL-10 regulatesgrowth and/or differentiation of B cells, NK cells, cytotoxic and helperT cells, mast cells, granulocytes, dendritic cells, keratinocytes, andendothelial cells. IL-10 plays a key role in differentiation andfunction of a newly appreciated type of T cell, the T regulatory cell,which may control of immune responses and tolerance in vivo.

The invention provides methods of inducing or enhancing IL-10production. A cell or tissue is contacted with a galectin-1 polypeptidein an amount sufficient to induce IL-10 production. A tissue is forexample tissue of the immune system such as lymph node tissue.Optionally, the tissue is inflamed. The cell is any cell capable ofproducing IL-10. The cell is non-cancerous cell. Alternatively, the cellis a cancerous cell. Preferably, the cell is an immune cell. Forexample, the cell is a lymphocyte such a B-cell, or a T-cell, adendritic cell, a monocyte or a macrophage. The cell is activated.Alternatively, the cell is non-activated. The cell is CD4 and/or CD8positive. The cell is contacted in vivo, in vitro or ex vivo.

The cell contacted with the composition produces a greater amount ofIL-10 production compared to a reference cell. A reference cell or cellpopulation has not been exposed to the composition. Preferably, thereference cell is similar to the cell exposed to the composition. Forexample, if the cell exposed to the composition is a non-activatedT-cell, the reference cell population comprised non-activated T-cell.Alternatively, the reference cell population is derived from a databaseof molecular information derived from cells for which the assayedparameter or condition is known.

Induction of IL-10 production is defined by an increase IL-10 expressionor activity. Enhancing IL-10 production is meant an increase of IL-10production compared to normal levels of IL-10 production. Expression ofIL-10 is determined at the RNA level using any method known in the art.For example, Northern hybridization analysis using probes whichspecifically recognize an IL-10 gene can be used to determine geneexpression. Alternatively, expression is measured using a quantitativereverse-transcription-based PCR assays, e.g., using primers specific forIL-10. IL-10 expression is also determined at the protein level, i.e.,by measuring the levels of IL-10 protein. Such methods are well known inthe art and include, e.g., immunoassays based on antibodies to IL-10 andcommercially available IL-10 screening assays.

Methods of Reducing Inflammation

Inflammation is inhibited by administering to tissue galectin-1polypeptide. Tissues to be treated include an intestinal tissue, acardiac tissue, a pulmonary tissue, a dermal tissue, or a hepatictissue. For example, the tissue is an epithelial tissue such as anintestinal epithelial tissue, pulmonary epithelial tissue, dermal tissue(i.e., skin), or liver epithelial tissue.

Inflammation is inhibited when one or more of a signs or symptoms ofinflammation is reduced compared to a tissue that has not been contactedwith a galectin-1 polypeptide. Signs and symptoms of inflammationinclude for example, redness, pain local warmth and/or swelling of thetreated tissue. Tissues are directly contacted with the polypeptides.Alternatively, the polypeptide is administered systemically. Galectin-1polypeptides are administered in an amount sufficient to decrease (e.g.,inhibit) immunosuppressive cytokine production. An immunosuppressivecytokine is a cytokine that reduces an inflammatory response. Forexample the immunosuppressive cytokine is IL-10. An inflammatoryresponse is evaluated morphologically by observing tissue damage,localized redness, raised temperature and swelling of the affected area.Alternatively, an inflammatory response is evaluated by measuringc-reactive protein, various cytokines (e.g., IL-1 in the tissue or inthe serum or plasma) or the presence of inflammatory cells. A decreasein white blood count generally indicates a decrease in inflammation.

Efficacy of treatment is determined in association with any known methodfor diagnosing or treating the immune mediated disorder. Alleviation ofone or more symptoms of the immune mediated disorder indicates that thecompound confers a clinical benefit.

The methods are useful to alleviate the symptoms of a variety of immunemediated disorders, such as an inflammatory disorder. The inflammatorydisorder is acute or chronic. Inflammatory disorders includecardiovascular inflammation, gastrointestinal inflammation, hepaticinflammatory disorders, pulmonary inflammation, autoimmune disease(e.g., systemic lupus erythematosus, multiple sclerosis, diabetes,dermatomyositis, polymyositis, inflammatory neuropathies (GuillainBarré, inflammatory polyneuropathies), vasculitis (Wegener'sgranulomatosus, polyarteritis nodosa), polymyalgia rheumatica, temporalarthritis, Sjogren's syndrome, Bechet's disease, Churg-Strauss syndrome,Takayasu's arteritis), neuroinflammatory disorders (e.g., multiplesclerosis), allergy (e.g., allergic rhinitis/sinusitis, skin allergiesand disorders (e.g., urticaria/hives, angioedema, atopic dermatitis,contact dermatitis, psoriasis), food allergies, drug allergies, insectallergies, mastocytosis, skeletal inflammation (e.g., arthritis,osteoarthritis, rheumatoid arthritis, spondyloarthropathies), andchronic and acute transplantation rejection.

The methods described herein lead to a reduction in the severity or thealleviation of one or more symptoms of an inflammatory disorder such asthose described herein. Inflammatory disorders are diagnosed and ormonitored, typically by a physician using standard methodologies

Gastrointestinal Inflammatory Disorders

Gastrointestinal inflammatory disorders include for example,inflammatory bowel disease, Crohn's Disease, colitis (i.e., ulcerative,ileitis or proctitis).

Ulcerative colitis is an inflammatory bowel disease that causesinflammation and sores, called ulcers, in the top layers of the liningof the large intestine. The inflammation usually occurs in the rectumand lower part of the colon, but it can affect the entire colon.Ulcerative colitis rarely affects the small intestine except for thelower section, called the ileum. Ulcerative colitis occurs most often inpeople ages 15 to 40, although children and older people develop thedisease. Ulcerative colitis affects men and women equally and appears torun in families. Crohn's Disease causes inflammation deeper within theintestinal wall. Crohn's disease usually occurs in the small intestine,but it can also occur in the mouth, esophagus, stomach, duodenum, largeintestine, appendix, and anus.

Symptoms of gastrointestinal inflammatory disorder are abdominal painand bloody diarrhea. Other symptoms include fatigue, weight loss, lossof appetite, rectal bleeding and loss of body fluids and nutrients.Gastrointestinal inflammation can also cause problems such as arthritis,inflammation of the eye, liver disease (fatty liver, hepatitis,cirrhosis, and primary sclerosing cholangitis), osteoporosis, skinrashes, anemia, and kidney stones.

Gastrointestinal inflammation is diagnosed using tests to check foranemia, which can indicate bleeding in the colon or rectum. In addition,a stool sample, can be taken to determine is there is bleeding orinfection in the colon or rectum. Alternatively, a colonoscopy isperformed to detect inflammation, bleeding, or ulcers on the colon wall.

Pulmonary Inflammatory Disorders

Pulmonary inflammatory disorders include for example, sinusitis acuterespiratory distress syndrome, asthma, bronchopulmonary dysplasia (BPD),emphysema, interstitial lung diseases, lung injury, and pulmonaryhypertension.

Asthma is a chronic lung condition that can develop at any age. It ismost common in childhood and occurs in approximately 7-10% of thepediatric population. Asthma affects twice as many boys as girls inchildhood; more girls than boys develop asthma as teenagers, and inadulthood, the ratio becomes 1:1 males to females. Symptoms of asthmainclude shortness of breath, wheezing, constriction of the chestmuscles, coughing, sputum production, excess rapid breathing/gasping,rapid heart rate and exhaustion. Asthma is diagnosed by physicalexamination, i.e. listening to the lungs with a stethoscope; examinationof nasal passages, chest x-ray, blood tests or spirometry.

Neuroinflammatory Disorders

Neuroinflammatory disorders include for example, multiple sclerosis,Parkinson's disease, Alzheimer's disease, and ischemic stroke.

Multiple sclerosis (MS) is a central nervous system inflammatorydemyelinating disease that is thought to have an autoimmunepathogenesis. MS is classified according to its clinical course intoseveral categories: benign, relapsing-remitting (the most commonvariant), progressive-relapsing, primary progressive and secondaryprogressive.

Pathologically, MS is characterized by the presence of areas ofdemyelination and T-cell predominant perivascular inflammation in thebrain white matter. Some axons may be spared from these pathologicalprocesses. Disease begins most commonly with acute or subacute onset ofneurological abnormalities. Initial and subsequent symptoms maydramatically vary in their expression and severity over the course ofthe disease, that usually lasts for many years. Early symptoms mayinclude numbness and/or paresthesia, mono- or paraparesis, doublevision, optic neuritis, ataxia, and bladder control problems. Subsequentsymptoms also include more prominent upper motor neuron signs, i.e.,increased spasticity, increasing para- or quardriparesis, vertigo,incoordination and other cerebellar problems, depression, emotionallability, abnormalities in gait, dysarthria, fatigue and pain.

Clinical observation, results of Magnetic Resonance Imaging (presence ofareas of demyelination in the CNS), spinal fluid examination (presenceof oligoclonal bands and/or elevated IgG index) and sometimes tests ofevoked potentials constitute the basis for diagnosis. Differentialdiagnosis for MS includes other demyelinating diseases of the nervoussystem, often of a viral or postinfectious origin. Among them areencephalomyelitis, transverse myelitis, as well as other immune-mediatedconditions, which affect CNS, such as sarcoidosis, systemic lupuserythematous, Vitamin B-12 deficiency, etc.

Skeletal Inflammatory Disorders

Skeletal Inflammatory disorders include for example, arthritis,osteoarthritis, rheumatoid arthritis, and spondyloarthropathies.Arthritis is inflammation of one or more joints, characterized byswelling, warmth, and redness of the overlying skin, pain andrestriction of motion. There are over 200 diseases that may causearthritis. Arthritis can be divided into two main categories: (1)non-inflammatory arthritis (2) inflammatory arthritis. Arthritis candevelop as a result of an infection such as gonorrhea or Lyme diseases.

Osteoarthritis arthritis (OA), also called degenerative arthritis occurswhen the cushioning cartilage in a joint breaks down. Weight bearingjoints including the lower back, hips, knees and feet are most commonlyaffected. Symptoms include pain and stiffness that are affected bychanges in weather-usually worsening in damp, cool, rainy weather.Knees-instability or buckling, especially with going down stairs. OA isdiagnosed by physical exam, blood tests and x-ray.

Ankylosing Spondylitis Arthritis is a chronic inflammatory disease ofthe spine that can result in fused vertebrae and rigid spine. Symptomsof ankylosing spondylitis generally appear in young adults as swellingand pain in the lower back. Children, generally boys, occasionally alsodevelop symptoms in their hips and knees. While beginning in the lowerback, the pain and stiffness will gradually move up through the spineand into the neck. Patients with ankylosing spondylitis typicallyexhibit five out of the following six symptoms, though the severity ofthose symptoms will vary greatly between patients, Onset of pain before35 years of age, Pain and early morning stiffness of the spine,improvement with movement, gradual onset of symptoms, symptoms lastlonger than three months, deep breathing may be restricted . Inaddition, most people with the disease also have a genetic marker knownas HLA-B27.

Autoimmune Disorders

The term “autoimmune disease” refers to a varied group of more than 80chronic illnesses that involve almost every human organ system. In allof these diseases, the underlying problem is similar—the body's immunesystem becomes misdirected, attacking the very organs it was designed toprotect. Autoimmune diseases affect connective tissue nerves, muscles,the endocrine system, and the gastrointestinal system. Autoimmunedisorders include for example lupus, rheumatoid arthritis, multiplesclerosis, myasthenia gravis, and type 1 diabetes

Type 1 diabetes (also called “insulin-dependent diabetes mellitus” or“juvenile diabetes”) is the severe insulin-requiring form of diabetes.It usually affects teens and young under-30 adults, but can affectinfants or children.). Symptoms of Type 1 diabetes are usually quitesevere, and rapidly arise over weeks or months. Common symptoms includethirst, excessive urination, hunger, weight loss and irritability.

Lupus includes, Systemic lupus erythematosus (SLE), Discoid lupuserythematosus (DLE), Drug-induced lupus and Neonatal lupus.

SLE is the most common type of lupus and affects many parts of the bodyincluding joints, skin, kidneys, lungs, heart, blood vessels, nervoussystem, blood, and brain. SLE usually develops in people between theages of 15 and 44 years, it can occur in childhood or later in life. Thesigns of SLE vary and there are usually periods of both illness andwellness (also called remission or having no symptoms). Some people havejust a few signs of the disease while others have more. Its symptoms caninclude, “butterfly” rash across the nose and cheeks, skin rashes onparts of the body exposed to the sun, sores in the mouth or nose,painful or swollen joints, fever, weight loss, hair loss, fatigue, chestpain when taking deep breaths, purple or pale fingers or toes from coldor stress, abdominal pain, kidney inflammation, headaches and paranoia.

Lupus is diagnosis by medical history along with a physical exam andspecial tests, helps the physician rule out other diseases that can beconfused with lupus, having 4 (or more) of the 11 symptoms of lupus, asdefined by the American College of Rheumatology and labs test such asthe Antinuclear antibody (ANA).

DLE affects just the skin. Its symptoms include a red, raised rash onthe face, scalp, or other parts of the body, sores in the mouth or nose.The rash may become thick and scaly and may last for days or years. DLEis diagnoses by histological examination of skin biopsies. Drug-inducedlupus is a reaction to some prescription medicines. The symptoms of aresimilar to SLE, except you don't have problems with your kidneys orcentral nervous system. It can take months to years of taking themedicine before symptoms appear. After you stop taking the drug, itcould take days, weeks, or months for symptoms to go away.

Neonatal lupus, while rare, some newborn babies of women with SLE orother immune system disorders get lupus. Babies with neonatal lupus mayhave a serious heart defect. About one-half of babies with neonatallupus are born with a heart condition.

Rheumatoid arthritis (RA) is a chronic disease that causes pain,stiffness, swelling, and limitation in the motion and function ofmultiple joints. If left untreated, or improperly treated, RA canproduce serious destruction of one or more joints which frequently leadsto permanent disability. Though the joints are the principal body partaffected by RA, inflammation can develop in other body organs as well.

Symptoms of RA include pain, stiffness, swelling, redness and difficultymoving the joints through a full range of motion. The stiffness seen inactive RA is typically worst in the morning and lasts anywhere from 1-2hours to the entire day. While RA can affect just about any joint, somejoints, especially those of the hands and feet, tend be involved morefrequently than others. Other symptoms that can occur in RA include lossof energy, low-grade fevers, loss of appetite, dry eyes and mouthproducing a condition known as Sjogren's and soft skin lumps in areassuch as the elbow and hands called rheumatoid nodules

RA is difficult to diagnose because it may begin gradually with subtlesymptoms. Many diseases, especially early on, can behave similarly toRA. The diagnosis of RA is based on the symptoms described and typicalphysical examination findings characterized by warmth, swelling and painin the joints. Additionally, certain laboratory abnormalities such asanemia (low red blood cells), a positive rheumatoid factor (an antibodyfound in approximately 80% of RA patients), and an elevated erythrocytesedimentation rate or “sed rate” (a blood test that in most patientswith RA tends to correlate with the amount of inflammation in thejoints) are commonly found in RA.

Hepatic Inflammatory Disorders

Hepatic inflammatory disorders include for example, hepatitis such viralhepatitis, bacterial hepatitis, autoimmune hepatitis, drug inducedhepatitis or alcoholic hepatitis. The incidence and severity ofhepatitis vary depending on many factors, including the cause of theliver damage and any underlying illnesses in a patient. Common riskfactors include intravenous drug use, Tylenol overdose (the dose neededto cause damage is quite close to the effective dose so be sure to becareful to take Tylenol only as directed), risky sexual behaviors,ingestion of contaminated foods, and alcohol use.

Symptoms of hepatitis include dark urine, loss of appetite fatigue,jaundice, abdominal pain, black stool. Hepatitis is diagnosed byphysical exam, liver function test, autoimmune marker and serology.

Cardiac Disorders

Cardiac inflammatory disorders include for example pericarditis,endocarditis, mycocarditis. Cardiac inflammation also includes aninflammation that results from an acute cardiac event such as amyocardial infarction. Cardiac inflammation is distinguished from othercardiac disorders in that inflammation is typically acute while otherdisorder such atherosclerosis inflammations are chronic. Atherosclerosisresults in the build up of deposits of fatty substances, cholesterol,cellular waste products, calcium and in the inner lining of an artery(i.e., plaque). In contrast, cardiac inflammation affects the muscletissue of the heart.

Pericarditis, is inflammation of the pericardium and is characterized bychest pain. Patients who have suffered a myocardial infarction oftendevelop pericarditis over subsequent days or weeks. Pericarditis isdiagnosed by elevated ST segments on an electrocardiogram.

Endocarditis is the inflammation of the endocardium and causes a widevariety of symptoms, particularly in the earlier stages of infection.Symptoms include fevers, chills, fatigue, weight loss, muscle aches, andsweating. Endocarditis is diagnoses by the presence of a heart murmur oran echocardiogram.

Myocarditis is the inflammation of the heart muscle. The symptoms ofmyocarditis include fever, chest pain, abnormal heat beats, fatigue andshortness of breath. Myocarditis is typically diagnosed by aendomyocardial biopsy.

Food Allergies

Food allergies include for example those for peanuts, nuts, cow milk anddairy products, fish and shell fish, certain fruits and vegetables,chocolate, beer or wine, nickel and wheat, and also pollen-associatedfood allergy. A special category is egg allergy, as the effects of thisspecific food allergy are extended in vaccination when vaccines raisedin chicken eggs are applied in egg- or chicken-allergic people.

The symptoms of food allergy included skin (e.g. atopic dermatitis,allergic contact dermatitis, eczema), gastrointestinal or respiratorymanifestations, and also anaphylaxis. Medication often includesantihistamines, systemic corticosteroids, epinephrine or respiratorytreatments such as inhaled albuterol.

Symptoms are often associated with allergen-specific Ig-E increases.Incidence and severity of reported incidents are rising as are thenumbers of foods incriminated.

Methods of Increasing Transplant Survival

The immune system responds to a transplant with B cell antibodies and Tcell lymphocytes, which can attack the new organ. In addition,chemokines play an essential role in regulating and co-ordinating theinfiltration of leucocytes into grafts. Chemokines are expressed inskin, liver, heart, and kidney grafts following initial engraftment,ischemic injury, viral infection, and acute and chronic rejection.

Transplant (i.e., graft) survival is increased by administering to asubject a composition comprising a galectin-1 polypeptide. Optionally,subject is further administered a composition including otherimmunosuppressive compounds such as, for example, azathioprine,corticosteroids, cyclosporin (and cyclosporin A), and FK506, or acombination of any of the foregoing. Alternatively, transplant survivalis increased by contacting an organ with a composition comprising agalectin-1 polypeptide. For example, prior to transplantation the organis perfused with a galectin-1 polypeptide.

The transplant is an allograft. Alternatively the transplant is axenograft. Such transplants include but are not limited to kidney,liver, skin, pancreas, cornea, or heart. The subject can be any mammal,e.g., a human, a primate, mouse, rat, dog, cat, cow, horse, pig.

A galectin-1 polypeptide is administered in a therapeutically effectivedosage regime to reduce, prevent or delay the incidence of graftrejection following the transplant. The treatment is administered priorto the subject receiving a transplant. Alternatively, treatment isadministered after a subject receives an transplant. Optionally,treatment is administered concomitantly to the subject receiving thetransplant. Treatment is administered over a select period of time. Forexample, treatment is administered 1, 2, 3, 4, 5, 6 or more days.Preferably, treatment is administered for 1, 2, 3, 4 or more weeks. Insome methods, a single dose of about 1 mg/kg of a galectin-1 polypeptideis administered about every other week, commencing immediately prior totransplantation and continuing until at least 8 weeks aftertransplantation. In other methods, the dose is 0.25-0.5 mg/kg, 1.5 mg/kgor a fixed unit dose of, e.g., 5 mg, 10 mg or 20 mg. Usually between 2and 5 doses, (e.g., 2, 3, 4 or 5) are administered over a period ofabout 2 weeks to 2 months in order to prevent (i.e., reduce theincidence of rejection episodes for a period of at least 2 or 3 butpreferably 6 or 12 months after transplantation. Alternatively, thegalectin-1 polypeptide can be administered daily, biweekly, weekly,every other week, monthly or at some other interval for 1 week, 2 weeks,4 weeks, 8 weeks, 3-6 months or longer. Optionally, the galectin-1polypeptide is administered after the physician suspects organrejection. Organ rejection is determined by methods know in the art. Forexample, organ rejection is indicated if the subjects blood creatininestarts to rise slowly after it has been stable for some time.

Transplant survival is increased by reducing, preventing or delaying therejection of the organ. Rejection means that the subject's immune systemrecognizes the transplant as foreign. Rejection is acute, e.g.hyperacute rejection. Alternatively, rejection is chronic. Acuterejection of a transplanted organ may occur within seconds or minutes ofexposing the organ to the recipient's circulation. Acute rejectionoccurs in the first few days (particularly the first few weeks) after atransplant. In contrast, chronic rejection is long-term and it startsslowly. The subjects immune system attacks and reject the transplant,but in a different way than in acute rejection. Chronic rejection lookslike a slow ageing of the transplanted organ. Chronic rejection usuallyoccurs more than a year after the transplant operation.

By survival rate of the transplant is meant the time before thetransplant is rejected by the subject. For example, survival isincreases when the transplant survives at least 1, 2, 4 or 8 weeks aftertransplant. Preferably, the transplant survives 3, 6, 13 months. Morepreferably, the transplant survives 2, 3, 5 or more years.

Methods of Vaccination

The invention provides a method of vaccination (i.e., immunization) of asubject. Specifically, the immune response to an antigen is improvedupon vaccination of a subject with a composition containing a galectin-1polypeptide. Galectin-1 treatment provides a positive stimulus for animmune reaction. A subject is immunized by administration to the subjecta composition containing a galectin-1 polypeptide and a compositioncontaining an antigen. An antigen is any compound to which a immuneresponse is desired. For example, an antigen is a protein, aglycoprotein, a lipoprotein, a polysaccharide, a lipopolysaccharide, alipid, glycolipid, a polynucleotide or a small molecule (e.g., ahapten). Optionally the antigen is linked, e.g., covalently linked to acarrier protein. The subject is at risk of developing or suffering froman infection, e.g., bacterial, viral or fungal. Infections include,Hepatitis C, HIV, Hepatitis B, Papilloma virus, Malaria, Tuberculosis,Herpes Simplex Virus, Epstein Barr Virus, Chlamydia, or Influenza.Alternatively, the subject is at risk of developing or suffering fromcancer. The cancer is for example breast, lung, colon, prostate,pancreatic, cervical cancer lymphoma or melanoma.

Vaccination is conducted by conventional methods. For example, thecomposition can be used in a suitable diluent such as saline or water,or complete or incomplete adjuvants. The vaccine can be administered byany route appropriate for eliciting an immune response such asintravenous, intraperitoneal, intramuscular, subcutaneous, and the like.The vaccine may be administered once or at periodic intervals until aimmune response is elicited. An immune response may be detected by avariety of methods known to those skilled in the art, including but notlimited to, detecting antigen specific antibodies, cytotoxicity assay,proliferation assay and cytokine release assays.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the overall seriousness of the diseaseor disorder, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Ultimately, the attendingphysician will decide the amount of protein of the present inventionwith which to treat each individual patient.

Methods of Inducing Apoptosis

Also included in the invention are methods of inducing apoptosis. In oneaspect apoptosis is induced in subject in need thereof by administeringa multimeric galectin-1 polypeptide in an amount sufficient to induceapoptosis. The subject can be e.g., any mammal, e.g., a human, aprimate, mouse, rat, dog, cat, cow, horse, pig. In various aspects thesubject is susceptible to cancer or an autoimmune disorder.

Apoptosis, also known as programmed cell death, plays a role indevelopment, aging and in various pathologic conditions. In developingorganisms, both vertebrate and invertebrate, cells die in particularpositions at particular times as part of the normal morphogeneticprocess. The process of apoptosis is characterized by, but not limitedto, several events. Cells lose their cell junctions and microvilli, thecytoplasm condenses and nuclear chromatin marginates into a number ofdiscrete masses. As the nucleus fragments, the cytoplasm contracts andmitochondria and ribosomes become densely compacted. After dilation ofthe endoplasmic reticulum and its fusion with the plasma membrane, thecell breaks up into several membrane-bound vesicles, apoptotic bodies,which are usually phagocytosed by adjacent bodies. As fragmentation ofchromatin into oligonucleotides fragments is characteristic of the finalstages of apoptosis, DNA cleavage patterns can be used as and in vitroassay for its occurrence (Cory, Nature 367: 317-18, 1994).

A multimeric galectin-1 polypeptide can be administered with ananti-angiogenic compound. Examples of an anti-angiogenic compoundinclude, but are not limited to, a tyrosine kinase inhibitor, anepidermal-derived growth factor inhibitor, a fibroblast-derived growthfactor inhibitor, a platelet-derived growth factor inhibitor, a matrixmetalloprotease (MMP) inhibitor, an integrin blocker, interferon alpha,interferon-inducible protein 10, interleukin-12, pentosan polysulfate, acyclooxygenase inhibitor, a nonsteroidal anti-inflammatory (NSAID), acyclooxygenase-2 inhibitor, carboxyamidotriazole, tetrahydrocortizol,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, endostatin, troponin-1, an antibody to VEGF,platelet factor 4 or thrombospondin.

The multimeric galectin-1 polypeptide can further be administered withan chemotherapeutic compound. Examples of chemotherapeutic compoundsinclude, but are not limited to, paclitaxel, Taxol, lovastatin,minosine, tamoxifen, gemcitabine, 5-fluorouracil (5-FU), methotrexate(MTX), docetaxel, vincristin, vinblastin, nocodazole, teniposide,etoposide, adriamycin, epothilone, navelbine, camptothecin,daunonibicin, dactinomycin, mitoxantrone, amsacrine, epirubicin oridarubicin.

In another aspect, apoptosis is induced in a cell by contacting a cellwith a multimeric galectin-1 polypeptide in an amount sufficient toinduce apoptosis. The multimeric galectin-1 polypeptide is stable. Thecell population that is exposed to, i.e., contacted with, the multimericgalectin-1 polypeptide can be any number of cells, i.e., one or morecells, and can be provided in vitro, in vivo, or ex vivo.

The amount of the stable multimeric galectin-1 polypeptide to induceapoptosis is in an amount less than a wild-type, e.g., monomericgalectin polypeptide. For example the cell is contacted or the subjectis administered the stable multimeric galectin-1 polypeptide at aconcentration less than 20 μM, 15 mM, 10 μM, 5 μM, 1, mM, 0.1 μM, or0.001 μM.

Some disease conditions are related to the development of a defectivedown-regulation of apoptosis in the affected cells. For example,neoplasias result, at least in part, from an apoptosis-resistant statein which cell proliferation signals inappropriately exceed cell deathsignals. Furthermore, some DNA viruses such as Epstein-Barr virus,African swine fever virus and adenovirus, parasitize the host cellularmachinery to drive their own replication. At the same time, theymodulate apoptosis to repress cell death and allow the target cell toreproduce the virus. Moreover, certain disease conditions such aslymphoproliferative conditions, cancer including drug resistant cancer,arthritis, inflammation, autoimmune diseases and the like may resultfrom a down regulation of cell death regulation. In such diseaseconditions, it would be desirable to promote apoptotic mechanisms.

Therapeutic Administration

The invention includes administering to a subject a compositioncomprising a galectin-1 polypeptide (referred to herein as “therapeuticcompound”).

An effective amount of a therapeutic compound is preferably from about0.1 mg/kg to about 150 mg/kg. Effective doses vary, as recognized bythose skilled in the art, depending on route of administration,excipient usage, and coadministration with other therapeutic treatmentsincluding use of other anti-inflammatory agents or therapeutic agentsfor treating, preventing or alleviating a symptom of a particularinflammatory disorder. A therapeutic regimen is carried out byidentifying a mammal, e.g., a human patient suffering from (or at riskof developing) an inflammatory disorder, using standard methods.

The pharmaceutical compound is administered to such an individual usingmethods known in the art. Preferably, the compound is administeredorally, rectally, nasally, topically or parenterally, e.g.,subcutaneously, intraperitoneally, intramuscularly, and intravenously.The compound is administered prophylactically, or after the detection ofan inflammatory event such as an asthma attack or an allergic reaction.The compound is optionally formulated as a component of a cocktail oftherapeutic drugs to treat inflammatory disorders. Examples offormulations suitable for parenteral administration include aqueoussolutions of the active agent in an isotonic saline solution, a 5%glucose solution, or another standard pharmaceutically acceptableexcipient. Standard solubilizing agents such as PVP or cyclodextrins arealso utilized as pharmaceutical excipients for delivery of thetherapeutic compounds.

The therapeutic compounds described herein are formulated intocompositions for other routes of administration utilizing conventionalmethods. For example, a multimeric galectin polypeptide is formulated ina capsule or a tablet for oral administration. Capsules may contain anystandard pharmaceutically acceptable materials such as gelatin orcellulose. Tablets may be formulated in accordance with conventionalprocedures by compressing mixtures of a therapeutic compound with asolid carrier and a lubricant. Examples of solid carriers include starchand sugar bentonite. The compound is administered in the form of a hardshell tablet or a capsule containing a binder, e.g., lactose ormannitol, a conventional filler, and a tableting agent. Otherformulations include an ointment, suppository, paste, spray, patch,cream, gel, resorbable sponge, or foam. Such formulations are producedusing methods well known in the art.

Therapeutic compounds are effective upon direct contact of the compoundwith the affected tissue. Accordingly, the compound is administeredtopically. For example, to treat contact dermatitis the compound isapplied to the area of skin affected. Alternatively, therapeuticcompound are administered systemically. Additionally, compounds areadministered by implanting (either directly into an organ such as theintestine, or liver or subcutaneously) a solid or resorbable matrixwhich slowly releases the compound into adjacent and surrounding tissuesof the subject.

For example, for the treatment of gastrointestinal inflammatorydisorders, the compound is systemically administered or locallyadministered directly into gastric tissue. The systemic administrationcompound is administered intravenously, rectally or orally. For localadministration, a compound-impregnated wafer or resorbable sponge isplaced in direct contact with gastric tissue. The compound or mixture ofcompounds is slowly released in vivo by diffusion of the drug from thewafer and erosion of the polymer matrix.

Inflammation of the liver (i.e., hepatitis) is treated for example byinfusing into the liver vasculature a solution containing the compound.Intraperitoneal infusion or lavage is useful to reduce generalizedintraperitioneal inflammation of prevent inflammation following asurgical event.

For the treatment of neurological inflammation the compound isadministered intravenously or intrathecally (i.e., by direct infusioninto the cerebrospinal fluid). For local administration, acompound-impregnated wafer or resorbable sponge is placed in directcontact with CNS tissue. The compound or mixture of compounds is slowlyreleased in vivo by diffusion of the drug from the wafer and erosion ofthe polymer matrix. Alternatively, the compound is infused into thebrain or cerebrospinal fluid using known methods. For example, a burrhole ring with a catheter for use as an injection port is positioned toengage the skull at a burr hole drilled into the skull. A fluidreservoir connected to the catheter is accessed by a needle or styletinserted through a septum positioned over the top of the burr hole ring.A catheter assembly (e.g., an assembly described in U.S. Pat. No.5,954,687) provides a fluid flow path suitable for the transfer offluids to or from selected location at, near or within the brain toallow administration of the drug over a period of time.

For treatment of cardiac inflammation, the compound is delivered forexample to the cardiac tissue (i.e., myocardium, pericardium, orendocardium) by direct intracoronary injection through the chest wall orusing standard percutaneous catheter based methods under fluoroscopicguidance for direct injection into tissue such as the myocardium orinfusion of an inhibitor from a stent or catheter which is inserted intoa bodily lumen. Any variety of coronary catheter, or a perfusioncatheter, is used to administer the compound. Alternatively the compoundis coated or impregnated on a stent that is placed in a coronary vessel.

Pulmonary inflammation is treated for example by administering thecompound by inhalation. The compounds are delivered in the form of anaerosol spray from pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

The invention will be further illustrated in the following non-limitingexamples.

EXAMPLE 1 General Methods

Culture Medium

The culture medium used throughout the experiments was RPMI 1640supplemented with 2 mM L-glutamine, 10% heat-inactivated FCS, 100 U/mlpenicillin, and 100 μg/ml streptomycin. In culture experiments with thegalectin-1 protein, the culture medium was additionally supplementedwith 1.2 mM DTT.

Preparation of recombinant human galectin-1

Recombinant human galectin-1 protein was prepared as follows. HumanLGALS1 DNA was amplified from human blood cDNA, using primers containinga NdeI or BamHI restriction site (GAL-1F: 5′-ggcatatggcttgtggtctggtcg-3′(SEQ ID NO:3), GAL-1R: 5′-ggggatcctcatcagtcaaaggcc-3′(SEQ ID NO:4)).After amplification, the PCR product was digested and ligated in theNdeI and BamHI site of the pET15b plasmid vector (Novagen, Madison,USA). The ligation mixture was transformed to Escherichia coli (E. coli)JM101 cells according to manufacturer's instructions. Plasmid DNA from aclone containing an insert of the expected size as determined byrestriction analysis was isolated, sequenced, and transformed to BL21Star (DE3) competent E. coli cells (Invitrogen, Paisley, UK), accordingto manufacturer's instructions.

For the construction of stable galectin-1 homodimers, we decided to usea FOS leucine zipper based construct. Between the FOS leucine zipper andgalectin-1, a hinge region was placed functioning as a flexible linker.The FOS leucine zipper was flanked by CGG and GGC amino acids at the N-and C-terminus respectively, to covalently link the zippers by disulfidebonds between cysteine residues (See above, SEQ ID NO: 1).

For galectin-1 production, transfected E. coli were grown in 2xTY mediumcontaining ampicillin in a 37° C. shaking incubator until OD₆₆₀˜0.8-1.0.IPTG (1 mM )was added and galectin-1 production was induced for 3 hours.Cells were harvested (15 minutes at 7500 g at 4° C.), lysed withextraction buffer containing 1 mg/ml lysozyme and extensively sonicated.The lysate was centrifuged and recombinant galectin-1 protein containinga His-tag was purified using TALON-beads (Clontech, Becton DickinsonBiosciences, Heidelberg, Germany) according to manufacturer'sinstructions. Galectin-1 protein was stored in buffer containing 20 mMTris (pH 8.0), 150 mM NaCl, 10% glycerol at −80° C., and used in allculture experiments in RPMI medium supplemented with 1.2 mM DTT.Recombinant human galectin-1 protein was routinely tested for mycoplasmaand endotoxins and these tests were consistently negative.

RNA isolation and semi-quantitative RT-PCR

Total RNA from cell pellets or frozen tissue sections was isolated usingthe Absolutely RNA RT-PCR Miniprep or Microprep kits (Stratagene, LaJolla, Calif.) according to manufacturer's instructions. 1-3 μg RNA wasreverse transcribed in a volume of 20 μl using random hexamers (300 ng)and Superscript II Reverse Transcriptase (Invitrogen) according tomanufacturer's instructions. PCR was performed in 60 μl with 1 unit ofTaq DNA polymerase (Amersham Pharmacia Biotech), the reaction bufferprovided by the manufacturer and 1 μl cDNA. PCR consisted of 20-40cycles of 30s 94° C., 30s 55° C. and 30s 72° C. The final extension stepconsisted of 7 minutes at 72° C. PCR samples were analyzed on a 1.5%agarose gel after an increasing number of PCR cycles. Primer used were:

LGALS1F 5′-cttgtggtctggtcgccag-3′(SEQ ID NO: 5), LGALS1R5′-tcgaaggtgatgcacacctc-3′ (SEQ ID NO: 6); GAPDHF5′-ccatcactgccactcagaagact-3′ (SEQ ID NO: 7), GAPDHR5′-ttactccttggaggccatgtagg-3′ (SEQ ID NO: 8). GAPDH was used as an RNAloading control. In each experiment, positive and negative controls wereincluded. Images were prepared using the Geldoc software (Bio-Rad,Veenendaal, The Netherlands) and in each case, inversed images areshown.

Real-Time PCR analysis for IL-10, IFN-γ, and HPRT

Primers (Invitrogen, Paisley, UK) and probes (Eurogentec, Seraing,Belgium) used for real-time PCR analysis were developed using primerdesign software. Primers (5′-3′) used were: IL-10F5′-atgaaggatcagctggacaactt-3′(SEQ ID NO: 9), IL-10R5′-ccttgatgtctgggtcttggt-3′ (SEQ ID NO: 10); IFN-γF5′-gaaacgagatgacttcgaaaagc-3′(SEQ ID NO: 11), IFN-γR5′-cgacctcgaaacagcatctg-3′(SEQ ID NO: 12); HPRTF5′-ggcagtataatccaaagatggtcaa-3′(SEQ ID NO: 13), HPRTR5′-gtctggcttatatccaacacttcgt-3′(SEQ ID NO: 14). Probe sequences labeled5′ with the FAM reporter dye and 3′ with the TAMRA quencher dyemolecules were: IL-10 5′-acctgggttgccaagccttgtctg-3′,IFN-γ5′-ccaagtgatggctgaactgtcgcc-3′, HPRT5′-caagcttgctggtgaaaaggacccc-3′. R(SEQ ID NO: 15) actions were performedin 384-wells plates (Applied Biosystems, the Netherlands) in a volume of20 μl containing real-time PCR mastermix (Eurogentec), 900 nM of eachprimer, and 200 nM of an individual probe. PCR amplifications wereperformed using the ABI prism 7900HT sequence detection system (AppliedBiosystems). Standard cycling conditions were used including apre-amplification step 50° C. for 2 minutes, 95° C. for 10 minutes,followed by an amplification of 45 cycles of 95° C. for 15 seconds and60° C. for 1 minute. All samples were analyzed in triplicate. Mean cyclethreshold values (Ct) and standard deviations (SD) were calculated forcytokine and housekeeping genes. The amount of cytokine target wasnormalized relative to the amount of housekeeping gene(ΔCt=Ct_(gene)−Ct_(HPRT)) and SD of the ΔCt (SD(ΔCt)) was calculated(SD(ΔCt)=√(SD_(gene))²+(SD_(HPRT))²). The relative amount of cytokinewas measured by determining the ΔΔCt(ΔΔCt=ΔCt_(test sample)−ΔCt_(calibrator)) and the factor difference iscalculated (2^(−ΔΔCt)). The range is given as 2^(−(ΔΔCt+SDΔCt)) and2^(−(ΔΔCt−SDΔCt)).

Patient selection and Immunohistochemistry

Renal graft material from patients with chronic (n=8) or acute rejection(n=7) was selected from the Tissue Bank at the Department of Pathology(Groningen, the Netherlands) (21). Control tissue was obtained from theunaffected part of nephrectomized kidneys from patients with renal cellcarcinoma (n=4) and unused donor kidneys (n=2).

TABLE 1 Patient Time No of Immunosuppression Immunosuppression CsAno^(a) Diagnosis of graft Transplants surveillance at nephrectomywithdrawal 2, 3, 5, 6 Normal — — — — — kidney  7 CR 3 m 1 Triple-MMFTriple-MMF —  8 CR 47 m 1 CP CP — 10 CR 29 m 1 Triple-Aza Triple-Aza —12 CR 16 m 1 Triple-MMF Aza-Pred 12 m 13 CR 61 m 1 Triple-Aza P20 1 m 14CR 3 m 1 Triple-MMF Triple-MMF — 15 HA 2 d 2 OKT3-Pred-Aza OKT3-Pred-Aza— 16 AR 7 d Triple-MMF Triple-MMF — 17 AR 1 d 2 ATG-MMF- PredATG-MMF-Pred — 19 AR 30 d 1 Triple-Aza Triple-Aza — 20 AR 12 d 1ATG-MMF-Pred ATG-MMF-Pred-CsA — 21 AR 34 d 1 Triple-Aza Aza-Pred 6 d^(a)Patient numbers are as described previously [20]. Normal kidneys -patients 2, 3: normal kidney adjacent to renal cell carcinoma; patients5, 6: unused donor kidney. HA, hyperacute rejection; AR, acuterejection; CR, chronic rejection; m, month; d, day. Aza,azathioprine; ATG, anti-thymocyte globulin; CP, cyclophosphamide; CsA,cyclosporin A; MMF, mycophenolate mofetil; OKT3, anti-CD3 antibody;Pred, prednisolone; P20, prednisolone 20 mg/day. Triple, treatment withcyclosporin A and prednisolone and the third immunosuppressive agent asindicated in the table.

Immunohistochemistry was performed according to standard procedures onfour-μm paraffin embedded tissue sections using a monoclonal antibodyagainst galectin-1 (clone 25C1, Novocastra, Newcastle upon Tyne, UK).

Galectin-1 treatment and ELISA

Peripheral blood mononuclear cells (PBMC) were obtained from healthyvolunteers and used directly for the galectin-1 experiments or used forthe isolation of CD4⁺ and CD8⁺ cells. CD4⁺ and CD8⁺T lymphocytes wereisolated by staining with fluorochrome-labeled 25 antibodies against CD3(anti-CD3-CyQ), CD4 (anti-CD4-F) and CD8 (anti-CD8-PE) (IQP, Groningen,the Netherlands), and cells were sorted on the MoFlo Cytometer(Cytomation, Fort Collins, Colo.). PBMC and sorted T cells werestimulated for 24 hours (1.10⁶ cells/ml) with anti-CD3 (10 ng/ml) oranti-CD3 plus anti-CD28, with or without different concentrations ofgalectin-1 protein, or galectin-1 protein alone. For inhibition assays,galectin-1 protein was pre-incubated for 30 minutes at room temperaturewith 0.1M lactose or polyclonal rabbit anti-galectin-1 sera (IQP,Groningen, the Netherlands) before addition to cells. IL-10 and IFN-γprotein productions were determined in cell-free culture supernatantscollected after 24 hrs stimulation, using ELISA (R&D systems, Oxon, UK)according to manufacturer's instructions. Multiple donors were analysedto confirm the results.

Alternatively, peripheral blood mononuclear cells (PBMC) were obtainedfrom healthy volunteers and cells were isolated by Ficoll-Hypaquecentrifugation. Isolated PBMC were stimulated for 6 or 24 hours in thepresence or absence of galectin-1 protein, with or without αCD3activation. For inhibition assays, galectin-1 protein was pre-incubatedfor 30 minutes at room temperature with 0.1M lactose before addition tothe cells. IL-10 and IL-1β protein productions were determined incell-free culture supernatants after 24 hours stimulation, usingcommercially available ELISA kits (R&D systems, Oxon, UK) according tomanufacturer's instructions. Multiplex ELISA for 6 cytokines (IFN-γ,IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12 and IL-13) was performed accordingto manufacturer's instructions (Biosource, Etten-Leur, The Netherlands).The Wilcoxon signed ranks test was used to determine the significance ofdifferences.

Measurement of Apoptosis

Apoptosis was measured using the phosphatidylserine detection kit (IQP,Groningen, The Netherlands). MOLT-4 T cells were cultured for 3 hours at37° C. (1.10⁶/ml in RPMI/10% FCS/1.2 mM DTT) in the presence or absenceof galectin-1 protein. After this, cells were adjusted to 0.1Mlactose/PBS and gently agitated for 10 minutes at room temperature todissociate galectin-1 from the cell membrane. Cells were washed withPBS, and resuspended in 110 μl calcium buffer (10 mM HEPES, 140 mM NaCl,2.5 mM CaCl₂, pH 7.4) containing 2.5 ul AnnexinV-FITC. Cells wereincubated for 20 minutes on ice, and washed once with calcium buffer.Cells were suspended in 160 μl calcium buffer containing 1 μl propidiumiodide (PI), incubated for 10 minutes on ice, and immediately analyzedby flow cytometry. For each sample 10,000 events were analyzed on aCoulter Epics-Elite flow Cytometer (Coulter Corporation, Hialeah, Fla.,USA). Data were analyzed using WinList 4.0 software (Verity SoftwareHouse Inc. Topsham, Me., USA).

Ex-vivo treatment of tissue biopsies by Galectin-1

Biopsies were taken according to standard procedures from the colon of apatient with colitis at 2 different locations (control (not inflamed)and inflamed tissue). The biopsies were incubated in culture medium andincubated for 8 hours at 37° C. with (0.5 and 2.5 μM) or without stablegalectin-1 protein. The biopsies were snap frozen in liquid nitrogenafter 8 hours and stored at −80° C. Frozen sections were used for RNAisolation to measure the IL-10 levels with qRT-PCR and forimmunohistology for T cells (CD3) and IL2receptor (T cell activation)according to standard procedures. Apoptosis was measured with the TUNELmethod according to the manufacturer's instructions (Roche Diagnostics,Almere, The Netherlands).

EXAMPLE 2 Galectin-1 Upregulates IL-10 Production in T Cells

To study the immunomodulatory effects of galectin-1, recombinantgalectin-1 was produced in E. coli and used in in vitro experiments.Galectin-1 exists in a reversible monomer-dimer equilibrium and, basedon the dissociation constant of 7 μM [6], the protein is predominantlypresent as a monomer at concentrations of <7 μM, or as a dimer atconcentrations of >7 μM. Since it is known that the form of the proteinis important in its function, the effect of different concentrations ofgalectin-1 protein on the production of different cytokines was studied.In total PBMC cultures, galectin-1 treatment caused a dose-dependentdownregulation of αCD3-induced IFN-γ production (FIG. 1A). The strongestdownregulation of IFN-γ production was observed using the highestgalectin-1 concentrations, which contains the highest concentration ofdimeric protein. Besides downregulation of IFN-γ, a marked anddose-dependent increase in IL-10 production was observed when PBMCs werecultured in the presence of high concentrations of galectin-1 protein(FIG. 1B). This galectin-1-induced IL-10 production could be inhibitedby pre-incubation with lactose or with anti-galectin-1 antibodies(rabbit polyclonal, IQP), with 62% and 41% inhibition respectively (FIG.1C).

Galectin-1 can bind to several T-cell surface glycoproteins like CD2,CD3, CD7, CD45, and CD43 [3-5,21]. To address whether specific T-cellsubsets were responsible for the IL-10 production following galectin-1treatment, FACS-sorted CD4+ and CD8+ T-cells from five independentdonors were stimulated with αCD3/αCD28 antibodies, with αCD3/αCD28 incombination with 20 μM galectin-1 protein, or with galectin-1 (20 μM)alone. As shown in FIG. 1D, CD4+ and CD8+ T-cells highly induced IL-10production following αCD3/αCD28 stimulation in combination with thedimeric form (20 μM) of the galectin-1 protein. No IL-10 was detectablein non-activated CD4+ and CD8+ T-cells (not shown), whereas incubationof cells with galectin-1 alone also resulted in upregulation of IL-10production (FIG. 1D and Table 2). In general, galectin-1-induced IL-10production was lower in non-activated cells than in αCD3/αCD28-activatedcells, but not significantly. In CD8+ T-lymphocytes, treatment withgalectin-1 also resulted in upregulation of IL-10 protein, although thelevels were lower than in CD4+ T-lymphocytes. Analysis ofgalectin-1-induced IL-10 production in sorted CD4+CD25+ T-cells revealedthat these cells only accounted for 0.15% of the IL-10 productionobserved in total PBMCs.

Besides the consistent upregulation of IL-10 following treatment withdimeric galectin-1, production of IFN-y was downregulated in CD4+ andCD8+ T cell subsets following αCD3/αCD28 stimulation in the presence ofhigh concentrations of galectin-1 compared to αCD3/αCD28 stimulationalone (p=0.043, FIG. 1E). Mean reduction of IFN-γ production was 36% inCD4+ T-cells and 49% in CD8+ T-cells.

Since galectin-1 treatment alone resulted in upregulation of IL-10production, the expression of the activation markers CD25 and CD69 wereinvestigated by FACS. Ad shown in FIG. 1F galectin-1 treatment resultedin a strong reduction of CD25 and CD69 positive leukocytes.

Table 3 summarizes the results obtained for IL-10 and IFN-γmRNA levelsfor five independent donors. In general, upregulation of IL-10 mRNAlevels and variable IFN-γ mRNA levels were observed. IL-10 mRNA wasupregulated in both CD4+ and CD8+ T-cells after treatment withgalectin-1, consistent with the ELISA results. IFN-γmRNA levels weresimilar or downregulated in CD4+ T-cells and similar or upregulated inCD8+ T-cells. Inconsistencies between IFN-γ protein and RNA levels(Tables 2 and 3) can be caused by measurement of protein and mRNA at thesame time point.

TABLE 2 Overview of IL-10 protein production in CD4+ and CD8+ T-cells of5 donors T-cell subset + treatment #1 #2 #3 4 # #5 CD4 + αCD3/αCD28 6265 165 240 539 CD4 + αCD3/αCD28 + Gal 588 263 616 456 1635 20 μM CD4 +Gal 20 μM 468 212 300 222 1699 CD8 + αCD3/αCD28 54 —^(a) 63 —^(a) 265CD8 + αCD3/αCD28 + Gal 268 71 239 62 709 20 μM CD8 + Gal 20 μM 238 134228 20 1139 ^(a)IL-10 levels below detection level of 15 pg/ml. TheIL-10 protein production (pg/ml) in sorted T-cells after 24 hstimulation as indicated. The Wilcoxon signed ranks test was used todemonstrate a significant induction of IL-10 in both CD4+ and CD8+T-cells treated with αCD3/αCD28 and galectin-1 compared to cells treatedwith αCD3/αCD28 alone (p = 0.043; p = 0.043).

TABLE 3 IL-10 and IFN-γmRNA in CD4+ and CD8+ T-lymphocytes of 5 donorsafter galectin-1 treatment T-cell subset + treatment # 1 #2 #3 #4 #5IL-10 factor^(a) CD4 + αCD3/αCD28 6.4 (5.9-6.9)^(b) 9.5 (8.3-11) 12(9.1-16) 13 (12-14) 5.8 (5.1-6.7) CD4 + αCD3/αCD28 + Gal 20 μM 24(19-30) 22 (20-25) 23 (19-28) 16 (15-17) 20 (18-22) CD4 + Gal 20 μM 65(45-94) 88 (73-105) 18 (15-22) 22 (17-28) 175 (163-188) CD8 + αCD3/αCD280.4 (0.2-0.6) 0.4 (0.3-0.6) 20 (19-21) 1.1 (1.0-1.3) 3.8 (3.6-3.9) CD8 +αCD3/αCD28 + Gal 20 μM 18 (17-19) 4.9 (4.0-6.0) 15 (13-18) 4.2 (3.7-4.7)22 (21-23) CD8 + Gal 20 μM 56 (44-72) 47 (41-54) 22 (17-29) 4.8(4.2-5.4) 210 (185-237) IFN-γfactor^(a) CD4 + αCD3/αCD28 23 (19-27) 217(200-235) 177 (144-218) 482 (458-507) 77 (72-83) CD4 + αCD3/αCD28 + Gal20 ∝M 7.0 (6.0-8.2) 170 (162-178) 131 (120-143) 572 (538-607) 118(111-126) CD4 + Gal20 ∝M —^(c) 2.9 (2.1-4.2) 1.6 (1.2-2.2) 1.5 (1.1-2.0)0.3 (0.2-0.3) CD8 + αCD3/αCD28 34 (25-45) 48 (43-55) 86 (73-101) 41(39-44) 135 (121-151) CD8 + αCD3/αCD28 + Gal 20 ∝M 36 (34- 36 (34-39)127 (121-134) 130 (118-143) 178 (159-199) 137 (132-142) 39) 127(121-134) 130 (118-143) 178 (159-199) 137 (132-142) CD8 + Gal 20 ∝M 2.6(1.9-3.6) 9.7 (6.8-14) 16 (14-20) 1.1 (0.8-1.4) 2.4 (1.9-2.9) ^(a)Factordifference calculated relative to cytokine production in a commoncalibrator (non-activated CD8+ T-cells), after normalization againstHPRT. ^(b)The ranges for the factor are given between parentheses andwere calculated by the formula: 2 − (_Ct + SD_Ct) and 2 − (_Ct − SD_Ct).^(c)Value below detection levels.

EXAMPLE 3 LGALS1 is Highly Expressed in Kidney Allograft Rejection

During allograft rejection, T cell responses play an important role. Totest if LGALS1 plays a role in the regulation of the immune responseduring allograft rejection, we tested LGALS1 expression in acute andchronic kidney allograft rejection samples. In all control kidneysexcept one, LGALS1 mRNA expression was very weak or absent, whereas inmost kidneys with chronic or acute allograft rejection, expression ofLGALS1 mRNA was highly upregulated (FIG. 2). IL-10 mRNA was present in ⅜cases with chronic rejection and not in the other cases with kidneyallograft rejection. Expression of IFN-γmRNA was observed in three caseswith chronic rejection and a weak signal was observed in three othercases of chronic rejection. No IFN-γmRNA expression was found in thecases with acute rejection and in the normal kidney cases (FIG. 2).However, the relative abundance of both cytokines was very low comparedto LGALS1 mRNA levels. (FIG. 2B)

Immunohistochemical staining for galectin-1 in normal kidney samplesshowed that the expression was mainly confined to glomerular mesangialepithelial cells and to smooth muscle cells of large vessels (FIG.3A-B). Occasionally cells in the interstitium stained positive forgalectin-1 (FIG. 3C), whereas in general no expression was observed inendothelial cells from control kidneys. During allograft rejection,galectin-1 expression in glomerular mesangial epithelial cells and insmooth muscle cells was similar to control kidneys (not shown). Inaddition, galectin-1 protein expression was strongly upregulated inendothelial cells from peritubular capillaries in the interstitium andin endothelial cells from large vessels in inflammatory regions (FIG.3D-F).

EXAMPLE 4 Evaluation of the Effect of Stable Galectin-1 HomodimerEffects

To test the efficiency of IL-10 induction of stable galectin-1homodimers (dGAL) in comparison with the wild-type galectin-1 protein(mGAL) we incubated PBMC with various concentration of both proteins.FIG. 5 demonstrates that the stable galectin-1 dimers can induce IL-10production in activated cells at a concentration up to 100 fold lessthat the wild-type galectin-1 protein. The same results were obtainedwith resting PBMC (results not shown). Pre-incubation of the galectin-1protein with lactose (galectin-1 inhibitor) indeed resulted in stronglyreduced levels of IL-10 production (FIG. 6). These data demonstrate thatthe stable galectin-1 dimers show a 100 fold enhanced activity in theinduction of IL-10 production.

To test the efficiency of apoptosis induction of stable galectin-1dimers (dGAL) in comparison with the wildtype galectin-1 protein (mGAL)we treated MOLT-4 T cells with various concentrations ranging from0.1-20 μM for mGAL and concentrations ranging from 0.1-5 μM for dGAL.FIG. 7 shows that mGAL induced apoptosis only at the highestconcentration of 20 μM as is shown by the percentage of AnnexinVpositive cells in comparison to untreated control cells. dGAL was 4-8fold more effective in the induction of apoptosis than mGAL. These datademonstrate that the stable galectin-1 homodimers also have enhancedactivity with respect to the induction of apoptosis.

EXAMPLE 5 Evaluation of the Effect of Stable Galectin Homodimers onCytokines

To test the potential of stable galectin-1 homodimers to modulate theproduction of other cytokines a multiplex ELISA were performed ongalectin-1 treated cells (24 hours) of 5 independent donors forcytokines reported to be modulated upon galectin-1 treatment. Theseanalyses revealed for dGAL no consistent changes for IFN-γ and IL-2 andno induction of IL-4, IL-5, IL-12 and IL-13. These results were similarto the effects observed with high concentrations of mGAL treatment. ForIL-10 a strong induction was observed in all five donors (FIG. 8).Analysis of the IL-10 mRNA levels confirmed the induction of high levelsof IL-10 mRNA at all three tested dGAL concentrations (FIG. 9). ForIL-1β a strong induction was observed which was most pronounced at thehighest concentration of the dGAL, whereas only a weak induction wasdetected at the 20 mM concentration of the mGAL protein (FIG. 10). ThisIL-1β induction was consistently present in all 5 donors (FIG. 11).Pre-incubation with lactose could efficiently block the induction ofIL-1β production (FIG. 12). These data demonstrate that a more effectiveinduction of IL-1β can be achieved with 0.2 mM of dGAL in comparison tothe highest concentration of the mGAL (20 mM). These data indicate thatstable galectin-1 homodimers are much more potent than the wild-typegalectin-1 protein.

EXAMPLE 6 Evaluation of ex-vivo Treatment of Biopsies with Galectin-1

Biopsies of a patient with IBD were treated ex-vivo with twoconcentrations of dGAL. The effect of galectin-1 on the non-inflamedcontrol biopsies was minimal when looking at the IL-10 mRNA levels (FIG.13) while the effect on the inflamed tissue was more pronounced. Aremarkable observation was that the effect of dGAL was more pronouncedat the lowest concentration indicating that it is essential to optimizethe most effective dGAL concentration.

Immunohistochemistry revealed no changes for the amount of infiltratingT cells and their activation pattern in the treated and untreatedbiopsies (results not shown). FIG. 11 shows a representative image of aTUNEL staining on a treated and untreated inflamed tissue biopsy. Thereis a significant increase in the amount of apoptotic cells in theinflamed biopsies after incubation with the dimeric galectin-1. In thecontrol tissues there is no increase in the amount of apoptotic cells incomparison to the untreated tissues (results not shown).

EXAMPLE 7 Evaluation of the Effect of Stable Galectin-1 Homodimer invivo

The effect of different concentrations of intravenous stable galectin-1homodimer treatment in healthy mice will be determined (same micestrains as used for the disease models). Composition and activation ofperipheral blood cells and spleen cell suspensions will be analyzed todetermine the effect of galectin-1 treatment. Three mouse model systemswill be used, IBD, psorisas and asthma model.

For all three models, stable galectin-1 homodimers will be given at twotime points, i.e. at induction of disease and at the time that firstsymptoms of established disease are present. In the first animalexperiments two different concentrations of stable galectin-1 treatmentwill be given intravenously. The efficiency of the treatment will becompared to saline treated animals and to IL-10 treated animals. Thiswill reveal the additional favorable effects of galectin-1 treatment ascompared to IL-10 treatment. To further establish the additional effectsof stable galectin-1 homodimers, galectin-1 treatment will be performedwith and without presence of neutralizing anti IL-10 antibodies.Comparison of these two groups of animals will gain insight in theeffects of galectin-1 itself and the secondary effects induced by thehigh amounts of IL-10 present in the affected tissue.

The final experiments will focus on the comparison of intravenousadministration of stable galectin-1 homodimers and local administrationof stable galectin-1 homodimers. This will be achieved with galectin-1tablets that dissolve in the small bowel for the IBD model; withgalectin-1 cream for the psoriasis model; and with a galectin-1 aerosolfor the asthma model. Again two different concentrations at twodifferent time points will be given to test the efficiency at the timepoint of disease induction and at established disease.

General Procedure IBD Model

Adoptive transfer of naive CD45RB^(hi) CD4⁺ T cells into immunodeficientmice leads to the development of a lethal wasting disease in therecipients with severe leukocyte infiltration in the colon accompaniedby marked epithelial hyperplasia (Powrie). Treatment of the mice withCD45RB^(low) CD4⁺ cells can inhibit the disease and IL-10 plays anessential role in this process (Asseman).

Sorted CD4⁺ CD45RB^(hi) spleen cells from BALB/C mice are intravenouslyinjected (4×10⁵ cells per mouse) in C.B-17 scid mice and mice areevaluated regularly for weight loss and condition of the stool. Three to5 weeks after the transfer of T cells the mice will start loosing weightand their stool will become soft. After 10 to 12 weeks they will havelost 15-20% of their body weight and some animals have to be sacrificeddue to their bad condition. In our model the animals will be sacrificedat 8 weeks. Colons will be measured and histology will be performed toevaluate the extent of the colitis and the effect of treatment (Leach).Lamina propria lymphocytes will be isolated and tested for theproduction of cytokines (IL-2, IFN-γ, TNF-α, IL-4 and IL-10) in ELISA's,subpopulations by flowcytometry and mRNA for cytokines and transcriptionfactors by quantitative PCR (Davenport). Immunohistological andmolecular techniques will be used to assess the extent of apoptosis.

Galectin-1 treatment will be given daily starting at day 0, to test theefficiency of galectin-1 stable homodimers to prevent the disease orstarting after 3 weeks, to test the efficiency of galectin-1 homodimertreatment to reduce the symptoms of established disease.

General Procedure Psoriasis Model

The IBD model can be expanded to a psoriasis model if on day 1 after Tcell transfer, the mice are injected with staphylococcal enterotoxin B(Davenport). This is a Th1 type psoriasis animal model comparable to theTh1 type T cells found in psoriasis patients (Schlaak).

Sorted CD4⁺CD45RB^(hi) spleen cells from BALB/C mice are intravenouslyinjected (4×10⁵ cells per mouse) in C.B-17 scid mice. After 1 day themice are injected intraperitoneally with 10 μg of staphylococcalenterotoxin B. The mice are evaluated for the presence and severity ofskin lesions in addition to weight and stool. Skin lesions startdeveloping after 3-4 weeks, and after 7 weeks the mice have 100%incidence of skin lesions. After 8 weeks animals are sacrificed andhistology is performed. Immunohistochemistry is performed to detectapoptotic cells and other changes in the effected tissue. Skininfiltrating lymphocytes are isolated via enzyme digestion. Isolatedlymphocytes are stimulated in vitro and cytokine (IL-2, IFN-γ, TNF-α,IL-4 and IL-10) productions are measured (Davenport).

Galectin-1 treatment will be given at day 0, to test the efficiency ofgalectin-1 stable homodimers to prevent the disease and after 3 weeks,to test the efficiency of galectin-1 homodimer treatment to reduce thesymptoms of established disease.

General Procedure Asthma Model

In vivo effects of galactin-1 treatment will be studied in a mouse modelfor asthma in which downregulatory effects of endogenous IL-10 onallergic airway inflammation were described (mr stampfli 1999 Am. J.Respir Cell Mol Biol). Asthma will be induced in female Balb/c mice,aged 8-10 wk, by daily exposure of aerosolized ovalbumin (1% wt/vol in0.9% saline) for 20 minutes over a period of 10 consecutive days. As acontrol, phosphate buffered saline (PBS) will be used. The aerosol willbe delivered to a perspex exposure chamber (9 liters) by a “De Vilbissnebulizer” (type 646, De Vilbiss, Somerset, Pa., USA) driven by anairflow of 8 L/Umin providing aerosol with an output of 0.33 m/min.

Twenty four hours after the last aerosol, acute airway obstruction afterOVA or PBS aerosol and airway hyper-responsiveness to methacholine willbe assessed in conscious, spontaneously breathing animals using awhole-body plethysmography system (Buxco Electronics, Petersfield, UK).Twenty four hours after assessment of bronchial hyperreactivity andovalbumin-induced airway obstruction, mice will be sacrificed.Bronchoalveolar lavage fluid will be used for assessment of cytokines(IL-4, IL-5, IL-10 and IL-13), and infiltrating inflammatory cells willbe isolated from the lung tissue for determination of airwayinflammation by flow cytometry (T cells, B cells, neutrophils,eosinophils and macrophages). In addition, a group of mice will besacrificed for immunohistochemical analysis of airway inflammation inthe lung. The percentage of apoptotic cells will be assessed in the lungtissue. During the asthma inducing procedure, serum will be taken atdays 0, 5 and 11 for determination of OVA-specific- or total serum IgElevels.

Galectin-1 treatment will be given at day 0, to test the efficiency ofgalectin-1 stable homodimers to prevent the disease and at day 4, totest the efficiency of galectin-1 homodimer treatment to reduce thesymptoms of established disease. Efficiency of galectin-1 treatment willbe based on reduction of acute airway obstruction, bronchialhyperreactivity and serum IgE levels. In addition we will study theinduction of IL-10 production in infiltrating cells and the percentageof apoptotic cells in the affected tissue.

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Other embodiments are within the following claims.

1. A method of decreasing inflammation of a tissue comprisingadministering to an inflamed tissue a composition comprising amultimeric galectin-1 polypeptide, wherein the said multimericgalectin-1 polypeptide is stable at a concentration of less than 7 μM.2. The method of claim 1, wherein said inflammation is chronic or acute.3. The method of claim 1, wherein said tissue is pulmonary tissue, livertissue, intestinal tissue, pancreatic tissue, lymphoid tissue or dermaltissue.
 4. The method of claim 1, wherein said intestinal tissue isintestinal epithelial tissue.
 5. The method of claim 1, wherein saidinflammation is skeletal inflammation, gastrointestinal inflammation,cardiovascular inflammation, pulmonary inflammation, or neurologicalinflammation.
 6. The method of claim 5, wherein said gastrointestinalinflammation is colitis.